Loop and stem dynamics during RNA hairpin folding and unfolding

Area of Science:

• Biophysics
• Molecular Biology
• Biochemistry

Background:

• 2-Aminopurine (2AP) is a fluorescent adenine analog used to study nucleic acid base stacking.
• RNA hairpin structures are crucial for various biological functions and their folding dynamics are complex.

Purpose of the Study:

• To investigate the folding thermodynamics and kinetics of an RNA hairpin using 2AP.
• To compare the contributions of loop and stem regions to RNA folding using 2AP fluorescence.
• To develop a free-energy landscape model for RNA hairpin folding.

Main Methods:

• Labeling of RNA hairpin (gacUACGguc) with 2AP at loop and stem positions.
• Monitoring RNA stability and folding/unfolding kinetics using 2AP fluorescence during thermal melts and laser temperature jumps.
• Performing molecular dynamics (MD) simulations (0.6 μsec) to analyze RNA conformations at different temperatures.

Main Results:

• Distinct thermodynamic and kinetic traces were observed for loop and stem labeling, yet both fit a common free-energy landscape.
• Differences in base stacking changes upon unfolding explain the observed variations between loop and stem probes.
• A minimal four-state free-energy landscape was proposed, validated by combined experimental and MD simulation data.
• A sequential folding model accurately approximates the full folding dynamics, with an initial frayed state being a heterogeneous ensemble.

Conclusions:

• 2AP serves as a reliable adenine substitution for mapping RNA structural element contributions to folding.
• The study proposes a comprehensive free-energy landscape and folding model for the studied RNA hairpin.
• The findings highlight the utility of combining fluorescence spectroscopy and MD simulations for elucidating RNA folding pathways.